U.S. Pat. No. 4,525,354 to Birch and Patil described a “non-peptide” antibiotic of M.W. “about 842” called “albicidin.” Albicidin is described as produced by culturing chlorosis-inducing strains of Xanthomonas albilineans isolated from diseased sugarcane, and mutants thereof. The antibiotic was isolated from the culture medium by adsorption on resin and was purified by gel filtration and High Performance Liquid Chromatography (HPLC). The chemical structure of this antibiotic was not determined and remained unknown, although the Birch and Patil patent disclosed spectral data for a fraction having antibiotic activity and the presence of approximately 38 carbon atoms and at least one COOH group.
Xanthomonas albilineans is a systemic, xylem-invading pathogen that causes leaf scald disease of sugarcane (interspecific hybrids of Saccharum species) (Ricaud and Ryan, 1989; Rott and Davis, 2000). Leaf scald symptoms include chlorosis, necrosis, rapid wilting, and plant death. Chlorosis-inducing strains of the pathogen produce several toxic compounds. The major toxic component, named albicidin, inhibits chloroplast DNA replication, resulting in blocked chloroplast differentiation and chlorotic leaf streaks that are characteristic of the plant disease (Birch and Patil, 1983, 1985b, 1987a and 1987b). Several studies established that albicidin plays a key role in pathogenesis and especially in the development of disease symptoms (Wall and Birch, 1997; Zhang and Birch, 1997; Zhang et al., 1999; Birch, 2001).
The prior art indicates that albicidin inhibits prokaryotic DNA replication and is bactericidal to a range of gram-positive and gram-negative bacteria (Birch and Patil, 1985a). Albicidin is therefore of interest as a potential clinical antibiotic (Birch and Patil, 1985a). However, low yield of toxin production in X. albilineans has slowed down studies into the chemical structure of albicidin and its therapeutic application (Zhang et al., 1998). The chemical structure of this albicidin remains unknown, however this albicidin has been partially characterized as a non-peptide antibiotic with a molecular weight of about 842 that contains approximately 38 carbon atoms with three or four aromatic rings, at least one COOH group, two OCH3 groups, a trisubstituted double bond and a CN linkage (Birch and Patil, 1985a; Huang et al., 2001).
Molecular cloning and characterization of the genes governing the biosynthesis of albicidin is of considerable interest because such information provides approaches to engineer overproduction of albicidin, to characterize its chemical structure, to allow therapeutic applications and to clarify the relationship between toxin production and the ability to colonize sugarcane. Two similar mutagenesis and complementation studies have been conducted to identify the genetic basis of albicidin production in X. albilineans strains isolated in two different geographical locations, Australia and Florida.
One study of X. albilineans strain LS155 from Australia revealed that genes for albicidin biosynthesis and resistance span at least 69 kb (Wall and Birch, 1997). Subsequently, three genes required for albicidin biosynthesis were identified, cloned and sequenced from two Australian strains of X. albilineans (LS155 and Xa13): xabA, xabB and xabC (Huang et al., 2001; Huang et al. 2000a, 2000b). The xabB gene encodes a large protein with a predicted size of 525.6 kDa, with a modular architecture indicative of a multi functional polyketide synthase (PKS) linked to a nonribosomal peptide synthetase (NRPS) (Huang et al., 2001). The xabC gene, located immediately downstream from xabB, encodes an S-adenosyl-L-methionine (SAM)-dependent O-methyltransferase (Huang et al., 2000a). The xabA gene, located in another region of the genome, encodes a phosphopantetheinyl transferase required for post-translational activation of PKS and NRPS enzymes (Huang et al., 2000b).
These first results demonstrated that the albicidin biosynthesis apparatus is a PKS and/or NRPS system. Such systems assemble simple acyl-coenzyme A or amino acid monomers to produce polyketides and/or nonribosomal peptides (Marahiel et al., 1997; Cane, 1997; Cane and Walsh, 1999). These metabolites form very large classes of natural products that include numerous important pharmaceuticals, agrochemicals, and veterinary agents such as antibiotics, immunosuppressants, anti-cholesterolemics, as well as antitumor, antifungal and antiparasitic agents. Genetic studies of prokaryotic PKS and NRPS produced detailed information regarding the function and the organization of genes responsible for the biosynthesis of polyketides and nonribosomal peptides. Such knowledge, in turn, made it possible to produce combinations of PKS and NRPS genes from different microorganisms in order to produce novel antibiotics (McDaniel et al., 1999; Rodriguez and McDaniel, 2001; Pfeifer et al., 2001). Investigating the complete albicidin biosynthesis apparatus is therefore of great interest because such results may contribute to the knowledge as to how PKS and NRPS interact and how they might be manipulated to engineer novel molecules.
A second study with X. albilineans strain Xa23R1 from Florida revealed that at least two gene clusters, one spanning more than 48 kb, are involved in albicidin production (Rott et al., 1996). This conclusion was based on the following data: (I) fifty Xa23R1 mutants defective in albicidin production were isolated; (ii) a Xa23R1 genomic library of 845 clones, designated pALB1 to pALB845, was constructed; (iii) two overlapping DNA inserts of approximately 47 kb and 41 kb, from clones pALB540 and pALB571 respectively, complemented forty-five mutants and were supposed to contain a major gene cluster involved in albicidin production; (iv) a DNA insert of approximately 36 kb, from clone pALB639, complemented four of the five remaining mutants not complemented by pALB540 and pALB571, and was supposed to contain a second region involved in albicidin production; and (v) the remaining mutant, AM37, which was not complemented by any of the three cosmids pALB540, pALB571 and pALB639, was supposed to be mutated in a third region of the genome involved in albicidin production.
The DNA sequences of all of the genes required to produce the albicidin family of polyketide antibiotics, the expressed protein amino acid sequences of all of the genes, and the deduced structure of Albicidin have not been previously reported, although fragmentary sequences that include three of the biosynthetic genes have been reported. Identification of one albicidin gene, xabC, as a methyltransferase gene involved in albicidin biosynthesis is reported by Huang, G., Zhang, L. & Birch, R. G. (2000a, Gene 255, 327-333) and claimed as biologically active in producing a polyketide antibiotic in PCT WO 02/24736 A1. Identification of a second albicidin gene, xabA, as a phosphopantetheinyl transferase gene is reported by Huang, G., Zhang, L. and Birch, R. G. (2000b) Gene 258, 193-199 and claimed as biologically active in producing a polyketide antibiotic in PCT WO 02/24736 A1. Huang, G., Zhang, L. & Birch, R. G. (2001) Microbiology 147, 631-642, report a DNA sequence of xabB (GenBank accession # AF239749), a multi functional polyketide-peptide synthetase that may be essential for albicidin biosynthesis in Xanthomonas albilineans. This xabB gene is reported as full length by Birch in PCT WO 02/24736 A1 (their seq. ID #1) and claimed by Birch in PCT WO 02/24736 A1 as a biologically active polyketide synthase of 4,801 amino acids in length, enabling production of albicidin. However, the DNA sequence reported by Huang et al. (2001) in GenBank AF239749 and by Birch in PCT WO 02/24736 A1 (their seq. ID #1) appears to be incomplete and missing 6,234 bp of DNA sequence encoding 2,078 amino acids. The subject invention provides the complete DNA sequence of xabB (albI, our seq. 20) as 20,637 bp, encoding a biologically active polyketide synthase of 6,879 amino acids of in this application (our seq ID #26). Factors affecting biosynthesis by Xanthomonas albilineans of albicidins antibiotics and phytotoxins are discussed in J. Appl. Microbiol. 85, 1023-1028. and Wall, M. K. & Birch, R. G. (1997). Genes for albicidin biosynthesis and resistance span at least 69 kb in the genome of Xanthomonas albilineans. Lett. Appl. Microbiol. 24, 256-260. A gene from X. albilineans strain Xa13, designed AlbF, which confers high level albicidin resistance in Escherichia coli and which encodes a putative albicidin efflux pump, was directly submitted to Genbank by Bostock and Birch (Accession No. AF403709).